|
| Status |
Public on Aug 14, 2012 |
| Title |
medium CcpA induction replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Net intensity (tr.mean) {A}
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: MP902
treatment: 0.002 μM aTc
reference: test
|
| Treatment protocol |
TY medium was supplemented with 0.2% xylose for induction of tetR expression and different concentrations of aTc for ccpA expression induction.
|
| Growth protocol |
B. subtilis strain MP902 [strain MP901 (Ptet-ccpA, KmR) carrying pWH119 (Pxyl-tetR, EmR)] was grown at 37°C (with shaking) in 50 ml of reach TY medium supplemented with 15 mg/ml kanamycine, 2.5 mg/ml arythromycine, 1% glucose, 0.2% xylose and different concentrations of anhydrotetracyclin (aTc): 0, 0.02, 0.002 or 0.0001 μM.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
16 ml of culture at OD600=0.8 was harvested for total RNA isolation. RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) according to the manufacturer's instruction. Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures and from one culture of each strain an additional sample was collected for technical replicate.
|
| Label |
Cy3
|
| Label protocol |
20 μg of total RNA was incubated for 16 h at 42°C in the presence of 400 U Superscript III RNase H reverse transcriptase (Invitrogen), 0.2 mM aminoallyl dUTP (Amersham), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, and 3.2 g random nonamers. The synthesized cDNA was then labeled by coupling either Cy3 or Cy5 to the dUTP aminoallyl reactive group (CyScribe postlabeling kit; Amersham Biosciences) and was purified using a CyScribe GFX purification kit (Amersham Biosciences).
|
| |
|
| Channel 2 |
| Source name |
Net intensity (tr.mean) {B}
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: MP902
treatment: 0 μM aTc
reference: control
|
| Treatment protocol |
TY medium was supplemented with 0.2% xylose for induction of tetR expression and different concentrations of aTc for ccpA expression induction.
|
| Growth protocol |
B. subtilis strain MP902 [strain MP901 (Ptet-ccpA, KmR) carrying pWH119 (Pxyl-tetR, EmR)] was grown at 37°C (with shaking) in 50 ml of reach TY medium supplemented with 15 mg/ml kanamycine, 2.5 mg/ml arythromycine, 1% glucose, 0.2% xylose and different concentrations of anhydrotetracyclin (aTc): 0, 0.02, 0.002 or 0.0001 μM.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
16 ml of culture at OD600=0.8 was harvested for total RNA isolation. RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) according to the manufacturer's instruction. Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures and from one culture of each strain an additional sample was collected for technical replicate.
|
| Label |
Cy5
|
| Label protocol |
20 μg of total RNA was incubated for 16 h at 42°C in the presence of 400 U Superscript III RNase H reverse transcriptase (Invitrogen), 0.2 mM aminoallyl dUTP (Amersham), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, and 3.2 g random nonamers. The synthesized cDNA was then labeled by coupling either Cy3 or Cy5 to the dUTP aminoallyl reactive group (CyScribe postlabeling kit; Amersham Biosciences) and was purified using a CyScribe GFX purification kit (Amersham Biosciences).
|
| |
|
| |
| Hybridization protocol |
The labelled cDNA was hybridized to oligo DNA microarray in Ambion Slidehyb #1 buffer (Ambion Eurpe Ltd) at 48°C for 18 hours. Next, microarray slides were washed in 2 × SSC with 0.5% SDS, 1 × SSC with 0.25% SDS and 1 × SSC with 0.1% SDS and dried by centrifugation.
|
| Scan protocol |
Images were acquired with GeneTac LS IV confocal laser scanner (Genomics Solutions).
|
| Description |
replicate 3
slide 28
|
| Data processing |
Dual-channel array images were analyzed with ArrayPro 4.5 software (Media Cybernetics Inc.). Spots were screened visually to identify those of low quality. Slide data were processed with MicroPreP (van Hijum et. al. (2003) Appl. Bioinformatics 2, 241-244). Net signal intensities were calculated by grid-based background subtraction. A grid-based Lowess transformation was performed for slide normalization, negative and empty values were removed, and outliers were removed by the deviation test. Expression ratios of overexpression mutant strain over the wild-type strain were calculated and are represented in Matrix1. Further analysis was performed with a Cyber-T Student t test for paired data and resulting ratios are represented in Matrix2 (provided as a supplementary file on the Series record).
Matrix2 value definition: log2 normalized across samples mean ratio representing test/control
|
| |
|
| Submission date |
Jan 17, 2012 |
| Last update date |
Aug 15, 2012 |
| Contact name |
Bogumila Marciniak |
| Organization name |
Rijksuniversiteit Groningen
|
| Department |
Department of Genetics
|
| Street address |
Nijenborgh 7
|
| City |
Groningen |
| ZIP/Postal code |
9747 AG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL15025 |
| Series (1) |
| GSE35154 |
High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis |
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